Conopeptides

ABSTRACT

The invention relates to novel conopeptides and/or novel Luses of conopeptides. The conopeptides of the invention are analogs of α-Conotoxin MII that are selective for α6-containing nAChRs as described herein.

ROSS-REFERENCE TO RELATED APPLICATION

The present application is claims the benefit of and priority under 35U.S.C. § 119(e) to U.S. Provisional Patent Application Ser. No.60/625,945, filed 9 Nov. 2004, incoroporated herein by reference.

This invention was made with Government support under Grants No.GM48677, MH53631, DA12242 and NS11323, awarded by the National Instituteof General Medical Sciences, National Institutes of Health, Bethesda,Md. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to novel conopeptides and/or novel uses ofconopeptides as described herein. More specifically, the presentinvention is directed to the conopeptide α-conotoxin MII analogs (α-MII)as described herein that are selective for α6-containing nicotinicacetylcholine receptors.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are referenced in the following text byauthor and date and are listed alphabetically by author in the appendedbibliography.

Conus is a genus of predatory marine gastropods (snails) whichenvenomate their prey. Venomous cone snails use a highly developedprojectile apparatus to deliver their cocktail of toxic conotoxins (alsoreferred to as conopeptides herein) into their prey. In fish-eatingspecies such as Conus magus the cone detects the presence of the fishusing chemosensors in its siphon and when close enough extends itsproboscis and fires a hollow harpoon-like tooth containing venom intothe fish. The venom immobilizes the fish and enables the cone snail towind it into its mouth via an attached filament. For general informationon Conus and their venom see the website addresshttp://grimwade.biochem.unimelb.edu.au/cone/referenc.html. Prey captureis accomplished through a sophisticated arsenal of peptides which targetspecific ion channel and receptor subtypes. Each Conus species venomappears to contain a unique set of 50-200 peptides. The composition ofthe venom differs greatly between species and between individual snailswithin each species, each optimally evolved to paralyse it's prey. Theactive components of the venom are small peptides toxins, typically12-30 amino acid residues in length and are typically highly constrainedpeptides due to their high density of disulphide bonds.

The venoms consist of a large number of different peptide componentsthat when separated exhibit a range of biological activities: wheninjected into mice they elicit a range of physiological responses fromshaking to depression. The paralytic components of the venom that havebeen the focus of recent investigation are the α-, ω- and μ-conotoxins.All of these conotoxins act by preventing neuronal communication, buteach targets a different aspect of the process to achieve this. Theα-conotoxins target nicotinic ligand gated channels, the μ-conotoxinstarget the voltage-gated sodium channels and the ω-conotoxins target thevoltage-gated calcium channels (Olivera et al., 1985; Olivera et al.,1990). For example a linkage has been established between α-, αA- &φ-conotoxins and the nicotinic ligand-gated ion chanuel; ω-conotoxinsand the voltage-gated calcium channel; μ-conotoxins and thevoltage-gated sodium channel; δ-conotoxins and the voltage-gated sodiumchannel; κ-conotoxins and the voltage-gated potassium channel;conantokins and the ligand-gated glutamate (NMDA) channel.

However, the structure and function of only a small minority of thesepeptides have been determined to date. For peptides where function hasbeen determined, three classes of targets have been elucidated:voltage-gated ion channels; ligand-gated ion chanmels, andG-protein-linked receptors.

Conus peptides which target voltage-gated ion channels include thosethat delay the inactivation of sodium channels, as well as blockersspecific for sodium channels, calcium channels and potassium channels.Peptides that target ligand-gated ion channels include antagonists ofNMDA and serotonin receptors, as well as competitive and noncompetitivenicotinic receptor antagonists. Peptides which act on G-proteinreceptors include neurotensin and vasopressin receptor agonists. Theunprecedented pharmaceutical selectivity of conotoxins is at least inpart defined by a specific disulfide bond frameworks combined withhypervariable amino acids within disulfide loops (for a review seeMcIntosh et al., 1998).

Due to the high potency and exquisite selectivity of the conopeptides,several are in various stages of clinical development for treatment ofhuman disorders. For example, two Conus peptides are being developed forthe treatment of pain. The most advanced is ω-conotoxin MVIIA(ziconotide), an N-type calcium channel blocker (see Heading, C., 1999;U.S. Pat. No. 5,859,186). ω-Conotoxin MVIIA, isolated from Conus magus,is approximately

1000 times more potent than morphine, yet does not produce the toleranceor addictive properties of opiates. ω-Conotoxin MVIIA has completedPhase III (final stages) of human clinical trials and has been approvedas a therapeutic agent. ω-Conotoxin MVIIA is introduced into humanpatients by means of an implantable, programmable pump with a catheterthreaded into the intrathecal space. Preclinical testing for use inpost-surgical pain is being carried out on another Conus peptide,contulakin-G, isolated from Conus geographus (Craig et al. 1999).Contulakin-G is a 16 amino acid O-linked glycopeptide whose C-terminusresembles neurotensin. It is an agonist of neurotensin receptors, butappears significantly more potent than neurotensin in inhibiting pain inin vivo assays.

In view of a large number of biologically active substances in Conusspecies it is desirable to further characterize them and to identifypeptides capable of treating disorders involving ion channels,ligand-gated channels, or receptors. Surprisingly, and in accordancewith this invention, Applicants have discovered novel conopeptides thatcan be useful for the treatment of disorders involving ion channels,ligand-gated channels, or receptors and could address a long felt needfor a safe and effective treatment.

SUMMARY OF THE INVENTION

The invention relates to novel conopeptides and/or novel uses ofconopeptides as described herein. More specifically, the presentinvention is directed to the conopeptide α-conotoxin Mll analogs((α-MII) as described herein that are selective for α6-containingnicotinic acetylcholine receptors.

The present invention is further directed to derivatives of theconopeptides described herein or pharmaceutically acceptable salts ofthese peptides. Substitutions of one amino acid for another can be madeat one or more additional sites within the described peptides, and maybe made to modulate one or more of the properties of the peptides.Substitutions of this kind are preferably conservative, i.e., one aminoacid is replaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example:alanine to glycine, arginine to lysine, asparagine to glutamine orhistidine, glycine to proline, leucine to valine or isoleucine, serineto threonine, phenylalanine to tyrosine, and the like.

These derivatives also include peptides in which the Pro residues may besubstituted by hydroxy-Pro (Hyp); the Glu residues may be substituted byγ-carboxyglutamate (Gla); the Arg residues may be substituted by Lys,omithine, homoargine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys,N,N,N-trimethyl-Lys or any synthetic basic amino acid; the Lys residuesmay be substituted by Arg, omithine, homoargine, nor-Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid;the Tyr residues may be substituted with meta-Tyr, ortho-Tyr, nor-Tyr,mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phosplho-Tyr, nitro-Tyr orany synthetic hydroxy containing amino acid; the Ser residues may besubstituted with Thr or any synthetic hydroxylated amino acid; the Thrresidues may be substituted with Ser or any synthetic hydroxylated aminoacid; the Phe residues may be substituted with any synthetic aromaticamino acid; the Trp residues may be substituted with Trp (D), neo-Trp,halo-Trp (D or L) or any aromatic synthetic amino acid; and the Asn,Ser, Tlir or Hyp residues may be glycosylated. The halogen may be iodo,radioiodo, chloro, fluoro or bromo; preferably iodo for halogensubstituted-Tyr and bromo for halogen-substituted Trp. The Tyr residuesmay also be substituted with the 3-hydroxyl or 2-hydroxyl isomers(meta-Tyr or ortho-Tyr, respectively) and corresponding O-sulpho- andO-phospho-derivatives. The acidic amino acid residues may be substitutedwith any synthetic acidic amino acid, e.g., tetrazolyl derivatives ofGly and Ala. The Met residues may be substituted with norleucine (Nle).The aliphatic amino acids may be substituted by synthetic derivativesbearing non-natural aliphatic branched or linear side chainsC_(n)H_(2n+2) up to and including n=8. The Leu residues may besubstituted with Leu (D). The Gla residues may be substituted with Glu.The N-terrninal Gln residues may be substituted with pyroGlu.

The present invention is further directed to derivatives of the abovepeptides and peptide derivatives which are acylic permutations in whichthe cyclic pennutants retain the native bridging pattern of nativetoxin. See Craik et al. (2001).

Examples of synthetic aromatic amino acid include, but are not limitedto, nitro-Phe, 4-substituted-Phe wherein the substituent is C₁-C₃ alkyl,carboxyl, hyrdroxymiethyl, sulphomethyl, halo, phenyl, —CHO, —CN, —SO₃Hand —NHAc. Examples of synthetic hydroxy containing amino acid, include,but are not limited to, such as 4-hydroxymnethyl-Phe,4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Examples ofsynthetic basic amino acids include, but are not limited to,N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala,2-[3-(2S)pyrrolininyl)-Gly and 2-[3-(2S)pyrrolininyl)-Ala. These andother synthetic basic amino acids, synthetic hydroxy containing aminoacids or synthetic aromatic amino acids are described in Building BlockIndex, Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containingamino acids and aromatic amino acids and pages 66-87 for basic aminoacids; see also http://www.amino-acids.com), incorporated herein byreference, by and available from RSP Amino Acid Analogues, Inc.,Worcester, Mass. Examples of synthetic acid amino acids include thosederivatives bearing acidic functionality, including carboxyl, phosphate,sulfonate and synthetic tetrazolyl derivatives such as described byOrnstein et al. (1993) and in U.S. Pat. No. 5,331,001, each incorporatedherein by reference, and such as shown il the following schemes 1-3.

Optionally, in the conopeptides of the present invention, the Asnresidues may be modified to contain an N-glycan and the Ser, Tlr and Hypresidues may be modified to contain an O-glycan (e.g., g-N, g-S, g-T andg-Hyp). In accordance with the present invention, a glycan shall meanany N-, S- or O-linked mono-, di-, tri-, poly- or oligosaccharide thatcan be attached to any hydroxy, amino or thiol group of natural ormodified amino acids by synthetic or enzymatic methodologies known inthe art. The monosaccharides making up the glycan can include D-allose,D-altrose, D-glucose, D-mannose, D-gulose, D-idose, D-galactose,D-talose, D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine(GlcNAc), D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose.These saccharides may be structurally modified, e.g., with one or moreO-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic acid,including combinations thereof. The gylcan may also include similarpolyhydroxy groups, such as D-penicillamine 2,5 and halogenatedderivatives thereof or polypropylene glycol derivatives. The glycosidiclinkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between theglycan and the amino acid may be alpha or beta, preferably alpha and is1-.

Core O-glycans have been described by Van de Steen et al. (1998),incorporated herein by reference. Mucin type O-linked oligosaccharidesare attached to Ser or Thr (or other hydroxylated residues of thepresent peptides) by a GaINAc residue. The monosaccharide buildingblocks and the linkage attached to this first GaINAc residue define the“core glycans,” of which eight have been identified. The type ofglycosidic linkage (orientation and connectivities) are defined for eachcore glycan. Suitable glycans and glycan analogs are described furtherin U.S. patent applicantion Ser. No. 09/420,797 filed 19 Oct. 1999 andin Intematioinal Patent Application No. PCT/US99/24380 filed 19 Oct.1999 (publication No. WO 00/23092), each incorporated herein byreference. A preferred glycan is Gal(β1→3)GalNAc(α1→).

Optionally, in the peptides of general formula I and the specificpeptides described above, pairs of Cys residues may be replaced pairwisewith isoteric lactam or ester-thioether replacements, such as Ser/(Gluor Asp), Lys/(Glu or Asp), Cys/(Glu or Asp) or Cys/Ala combinations.Sequential coupling by known methods (Barnay et al., 2000; Hruby et al.,1994; Bitan et al., 1997) allows replacement of native Cys bridges withlactam bridges. Thioether analogs may be readily synthesized usinghalo-Ala residues commercially available from RSP Amino Acid Analgues.In addition, individual Cys residues may be replaced with homoCys,seleno-Cys or penicillamine, so that disulfide bridges may be formedbetween Cys-homoCys or Cys-penicillamine, or homocys-penicllamine andthe like.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show that H9A and L15A analogs of α-MII discriminate betweenα6/α3β2β3 and α3β2 nAChRs. Rat nAChR subunits were heterologouslyexpressed in X. laevis oocytes. Concentration-response analysis of thepeptide block of ACh-induced current was performed as described in theExamples. FIG. 1A: α-Conotoxin MII blocked α3β2 and α6/α3β2β3 with IC₅₀values of 2.18 and 0.39 nM, respectively. See also Table 1 forconfidence intervals. The Hill slopes were 0.75±0.13 and 0.53±0.04,respectively. FIG. 1B: MII[H9A] blocked α3β2 and α6/α3β2β3 nAChRs withIC₅₀ values of 59.0 and 0.79 nM, respective, and with Hill slopes of0.83±0.08 and 0.73±0.08. FIG. 1C: MII[L15A] blocked α3β2 and α6/α3β2β3nAChRs with IC₅₀ values of 34 and 0.92 nM, respectively, and with Hillslopes of 0.58±0.08 and 0.75±0.08, respectively. The data are from threeto six separate oocytes; ±value is the standard error of the mean.

FIGS. 2A and 2B show the concentration-response analysis of α-conotoxinMII[E11A] on nAChR subtypes expressed in X. laevis oocytes. Peptide wasperfusion-applied at concentrations >100 nM and bath-applied at higherconcentrations as described in the Examples. FIG. 2A: block by MII[E11A]of β02-containing nAChRs. FIG. 2B: block by MII[E11A] of β04-containingand α7 nAChRs. Data are from three to five oocytes. Error bars areS.E.M. Results are summarized in Table 2. Note the strong preference forα6/α3*nAChRs.

FIGS. 3A and 3B show that The [H9A,L15A] analog of α-MII discriminatesbetween α6* and α3*nAChRs. (The * indicates the possible presence ofadditional subunits.) Peptide was applied to oocytes expressing theindicated nAChR subunit combinations as described in the Examples. FIG.3A: the peptide blocked rat α3β2 with an IC₅₀ of 4.8 μM (CI=3.5-6.6 μM)and n_(H) of 0.48±0.04. The peptide blocked rat α6/α3β2β3 with an IC₅₀of 2.4 nM (CI=1.7-3.4 nM) and n_(H) of 0.72±0.09. FIG. 3B: the peptideblocked rat α3β4 with an IC₅₀ of 7.8 μM (CI=5.3-11.5 μM) and n_(H) of0.75±0.1. The peptide blocked rat α6β4 with an IC₅₀ of 269 nM(CI=153-476 nM) and n_(H) of 0.60±0.09; ±values are standard error ofthe mean.

FIG. 4 shows the kinetics of block. MII, MII[H9A], MII[L15A], andMII[H9A;L15A] were applied to X. laevis oocytes heterologouslyexpressing rat α6/α3β2β3 and α3β2 nAChRs. Peptide at the indicatedconcentrations was bath-applied for 5 min and then washed out. Kineticsof unblock were monitored by applying a 1-s pulse of ACh every 1 min.

FIG. 5 shows the effects of MII[H9A,L15A] on additional nAChR subtypes.Peptide at 100 nM (α6/α3β2β3) and 1 μM (all other subtypes) wasbath-applied for 5 min to X. laevis oocytes expressing the indicated ratnAChR subunits. Traces are representative of experiments on three tofive oocytes. C, control response to ACh. Second response in each tracepair is the response to ACh in the presence of peptide.

FIG. 6 shows the concentration-response analysis of α-conotoxin MIIanalogs on native nAChRs. Analogs were assessed for their ability todisplace [l¹²⁵I]α-conotoxin MII binding on mouse brain homogenates asdescribed in the Examples. Nonspecific binding was defined with 1 μMepibatidine. K_(i) values are shown in Table 5. The Hill slope was0.95±0.13, 0.89±0.14, and 1.1±0.14 for MII[H9A], MII[L15A], and MII[H9A;L15A], respectively. ±values are standard enror of the mean. Data arefrom three to seven experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention is directed to novel conopeptides and/or noveluses of conopeptides as described herein. More specifically, the presentinvention is directed to the conopeptide α-conotoxin MII analogs (α-MII)as described herein that are selective for α6-containing nicotinicacetylcholine receptors.

The sequence for α-conotoxin MII and the (α-conotoxin MII analogsdescribed herein are set forth in Table A. TABLE A Analogs ofα-Conotoxin MII Sequence (SEQ ID NO) Calculated Observed Methodα-Conotoxin MII GCCSNPVpb CHLEHSNLC* (1) 1710.66 1711.0 MALDI Analog:S4A GCCANPVCHLEHSNLC* (2) 1694.67 1694.7 LSIMS N5A GCCSAPVCHLEHSNLC* (3)1667.65 1667.6 LSIMS P6A GCCSNAVCHLEHSNLC* (4) 1684.64 1684.6 LSIMS V7AGCCSNPACHLEHSNLC* (5) 1682.63 1682.7 MALDI H9A GCCSNPVCALEHSNLC* (6)1644.64 1644.6 MALDI L10A GCCSNPVCHAEHSNLC* (7) 1668.61 1668.6 MALDIE11A GCCSNPVCHLAHSNLC* (8) 1652.65 1652.6 MALDI H12A GCCSNPVCHLEASNLC*(9) 1644.64 1644.7 MALDI S13A GCCSNPVCHLEHANLC* (10) 1694.67 1694.6LSIMS N14A GCCSNPVCHLEHSALC* (11) 1667.65 1667.6 LSIMS L15AGCCSNPVCHLEHSNAC* (12) 1668.61 1668.6 MALDI H9A; L15A GCCSNPVCALEHSNAC*(13) 1602.59 1602.6 MALDI L10A; L15A GCCSNPVCHAEHSNAC* (14) 1626.571626.6 MALDI E11A; L15A GCCSNPVCHLAHSNAC* (15) 1610.61 1610.6 MALDI S4A;H9A GCCANPVCALEHSNLC* (16) 1628.64 1628.69 MALDIMALDI, matrix-assisted laser desorption ionization time-of-flight massspectrometryLSIMS, liquid secondary ionization mass spectrometry,; *, amidatedC-terminus.

Neuronal nicotinic acetylcholine receptors (nAChRs) activated by theendogenous neurotransmitter acetylcholine belong to the superfamily ofligand-gated ion channels that also includes GABA_(A),5-hydroxytryptamine-3, and glycine receptors (Changeux, 1993). Thesedifferent ligand-gated ion channels show considerable sequence andstructural homology. Each of the subunits has a relatively hydrophilicamino terminal half (˜200 amino acids) that constitutes an extracellulardomain. This is followed by three hydrophobic- transmembrane domains, alarge intracellular loop, and then a fourth hydrophobic transmembranespan.

A large number of genes have been cloned that encode subunits of nAChRs.It has been proposed that these subunits may be divided into subfamilieson the basis of both gene structure and mature protein sequence. Thesubunits α2, α3, α4, and α6 belong to subfamily III, tribe 1; β2 and β4belong to tribe III -2; and the putative structural subunits α5 and β3belong to tribe III-3 (Corringer et al., 2000). Within tribe III-1,subunits α3 and α6 show considerable sequence identity (˜80% in theligand-binding extracellular domain). Thus, designing ligands todistinguish between α3*and α6*is particularly challenging.

α-Conotoxin MII is a 16 amino acid peptide originally isolated from thevenom of the marine snail Conus magus. This peptide potently targetsneuronal in preference to the muscle subtype of nicotinic receptor withhigh affinity for both α3β2 and α6* nAChRs. Unfortunately, α-conotoxinMII may not distinguish well between α3* and α6*nAChRs (Kuryatov et al.,2000). In an effort to remedy this situation and produce a selectiveligand for α6* nAChRs, a series of α-conotoxin MII analogs have beengenerated as described herein.

The α6 subunit is expressed in catecholaminergic neurons and in retina(Le Novere et al., 1996, 1999; Vailati et al., 1999). In striatum, α6*nAChRs seem to play a central role in the modulation of dopaminerelease. Recently, homozygous null mutant (α6−/−) mice were generated.Receptor autoradiography studies in these animals indicate that the α6nAChR subunit is a critical component of [¹²⁵I]α-conotoxin MII bindingin the central nervous system (Champtiaux et al., 2002). Studies usingmice with nAChR subunit deletion indicate that α3 does not participatein most [¹²⁵I]α-conotoxin MII binding sites but does influenceexpression in the habenulo-peduncular tract (Whiteaker et al., 2002).Thus, α6-selective ligands is useful to distinguish the α6* majorityform from the α3* minority of such sites.

More specifically, Neuronal nicotinic acetylcholine receptors (nAChRs)both mediate direct cholinergic synaptic transmission and modulatesynaptic transmission by other neurotransmitters. Novel ligands areneeded as probes to discriminate among structurally related nAChRsubtypes. α-Conotoxin MII, a selective ligand that discriminates among avariety of nAChR subtypes, fails to discriminate well between somesubtypes containing the closely related α3 and α6 subunits.Structure-fuinction analysis of α-conotoxin MII was performed in anattempt to generate analogs with preference for α6-containing [α6*(asterisks indicate the possible presence of additional subunits)]nAChRs. Alanine substitution resulted in several analogs with decreasedactivity at α3* versus α6* nAChRs heterologously expressed in Xenopuslaevis oocytes.

From the initial analogs, a series of mutations with two alaninesubstitutions was synthesized. Substitution at His9 and Leu15(MII[H9A;L15A]) resulted in a 29-fold lower IC₅₀ at α6β4 versus α3β4nAChRs. The peptide had a 590-fold lower IC₅₀ for α6/α3β2 versus α3β2and a 2020-fold lower IC₅₀ for α6/α3β2β3 versus α3β2 nAChRs.MII[H9A;L15A] had little or no activity at α2β2, α2β4, α3β4, α4β2, α4β4,and α7 NAChRs. Functional block by MII[H9A;L15A] of rat α6/α3β2β3 nAChRs(IC₅₀=2.4 nM) correlated well with the inhibition constanit ofMII[H9A;L15A] for [¹²⁵I]α-conotoxin MII binding to putative α6β2* nAChRsin mouse brain homogenates (K_(i)=3.3 nM). Thus, structure-fuLnctionanalysis of α-conotoxin MII enabled the creation of novel selectiveantagonists for discriminating among nAChRs containing α3 and α6subunits. Although the conopeptides of the present invention can beobtained by purification from cone snails, because the amounts ofpeptide obtainable from individual snails are very small, the desiredsubstantially pure peptides are best practically obtained incommercially valuable amount by by chemical synthesis using solid-phasestrategy. For example, the yield from a single cone snail may be about10 micrograms or less of peptide. By “substantially pure” is meant thatthe peptide is present in the substantial absence of other biologicalmolecules of the same type; it is preferably present in an amount of atleast about 85% purity and preferably at least about 95% purity

The peptides of the present invention can also be produced byrecombinant DNA techniques well known in the art. Such techniques aredescribed by Sambrook et al. (1989). A gene of interest can be insertedinto a cloning site of a suitable expression vector by using standardtechniques. These techniques are well known to those skilled in the art.The expression vector containing the gene of interest may then be usedto transfect the desired cell line standard transfection techniques suchas calcium phosphate co-precipitation, DEAE-dextran transfection orelectroporation may be utilized. A wide variety of host/exprressionvector combinations may be used to express a gene encoding a conotoxinpeptide of interest. Such combinations are well known to a skilledartisan. The peptides produced in this manner are isolated, reduced ifnecessary, and oxidized, if necessary, to form the correct disulfidebonds.

One method of fornling disulfide bonds in the peptides of the presentinvention is the air oxidation of the linear peptides for prolongedperiods under cold room temperatures or at room temperature. Thisprocedure results in the creation of a substantial amount of thebioactive, disulfide-linked peptides. The oxidized peptides arefractionated using reverse-phase high performance liquid chromatography(HPLC) or the like, to separate peptides having different linkedconfigurations. Thereafter, either by comparing these fractions with theelutioll of the native material or by using a simple assay, theparticular fraction having the correct linkage for maximum biologicalpotency is easily determined. However, because of the dilution resultingfrom the presence of other fractions of less biopotency, a somewhathigher dosage may be required.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which constituent aminoacids are added to the growing peptide chain in the desired sequence.Use of various coupling reagents, e.g., dicyclohexylcarbodiimide ordiisopropylcarbonyldimidazole, various active esters, e.g., esters ofN-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavagereagents, to carry out reaction in solution, with subsequent isolationand purification of intermediates, is well known classical peptidemethodology. Classical solution synthesis is described in detail in thetreatise, “Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden,” (1974). Techniques of exclusively solid-phase synthesis areset forth in the textbook, “Solid-Phase Peptide Synthesis,” (Stewart andYoung, 1969), and are exemplified by the disclosure of U.S. Pat. No.4,105,603 (Vale et al., 1978). The fragment condensation method ofsynthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Otheravailable syntheses are exemplified by U.S. Pat. No. 3,842,067 (1974)and 3,862,925 (1975). The synthesis of peptides containing-carboxyglutamic acid residues is exemplified by Rivier et al. (1987),Nishiuchi et al. (1993) and Zhou et al. (1996).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups that will prevent a chemical reaction from occurring at that siteuntil the group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the α-amino protecting group to allow subsequent reaction to takeplace at that location. Accordingly, it is common that, as a step insuch a synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in its desired sequence in thepeptide chain with appropriate side-chain protecting groups linked tovarious ones of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting groLupshould be stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology. However, when peptides are not soprepared, they are preferably prepared using the Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin or paramethylbenzhydrylamine(MBHA) resin. Preparation of the hydroxyniethyl resin is described byBodansky et al. (1966). Chloromethylated resins are commerciallyavailable from Bio Rad Laboratories (Richmond, Calif.) and from Lab.Systems, Inc. The preparation of such a resin is described by Stewartand Young (1969). BHA and MBHA resin supports are commerciallyavailable, and are generally used when the desired polypeptide beingsynthesized has an unsubstituted amide at the C-terminus. Thus, solidresin supports may be any of those known in the art, such as one havingthe formulae —O—CH₂-resiln support, —NH BHA resin support, or —NH-MBHAresin support. When the unsubstituted amide is desired, use of a BHA orMBHA resin is preferred, because cleavage directly gives the amide. Incase the N-methyl amide is desired, it can be generated from an N-methylBHA resin. Should other substituted amides be desired, the teaching ofU.S. Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or shouldstill other groups than the free acid be desired at the C-terminus, itmay be preferable to synthesize the peptide using classical methods asset forth in the Houben-Weyl text (1974).

The C-terminal amino acid, protected by Boc or Fmoc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in Horiki etal. (1978), using KF in DMF at about 60° C. for 24 hours with stirring,when a peptide having free acid at the C-terminus is to be synthesized.Following the coupling of the BOC-protected amino acid to the resinsupport, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCI indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder & Lubke (1965).

After removal of the (α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. Selection of an appropriate coupling reagentis within the skill of the art. Particularly suitable as a couplingreagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU inthe presence of HoBt or HoAt).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder &Lubke (1965) and Kapoor (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF):CH₂Cl₂ (1:1)or in DMF or CH₂Cl₂ alone. In cases where intermediate coupling occurs,the coupling procedure is repeated before removal of the (α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by Kaiser et al. (1970). Coupling reactions can beperformed automatically, as on a Beckman 990 automatic synthesizer,using a program such as that reported in Rivier et al. (1978).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride or TFA for cleaving, one or more scavengers such asanisole, cresol, dimethyl sulfide and methylethyl sulfide are includedin the reaction vessel.

Cyclization of the linear peptide is preferably affected, as opposed tocyclizincg the peptide while a part of the peptido-resin, to createbonds between Cys residues. To effect such a disulfide cycliziniglinkage, fully protected peptide can be cleaved from ahydroxymetlhylated resin or a chloromethylated resin support byammonolysis, as is well known in the art, to yield the fully protectedamide intermediate, which is thereafter suitably cyclized anddeprotected. Alternatively, deprotection, as well as cleavage of thepeptide from the above resins or a benzhydrylamine (BHA) resin or amethylbenizlhydrylamine (MBHA), can take place at 0° C. withhydrofluoric acid (HF) or TFA, followed by oxidation as described above.

The peptides are also synthesized using an automatic synthesizer. Aminoacids are sequentially coupled to an MBHA Rink resin (typically 100 mgof resin) beginning at the C-terminus using an Advanced Chemtech 357Automatic Peptide Synthesizer. Couplings are carried out using1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluoroplhosphate (HBTU) and diethylisopro- pylethylamine (DIEA). TheFMOC protecting group is removed by treatment with a 20% solution ofpiperidine in dimethylfonnamide(DMF). Resins are subsequently washedwith DMF (twice), followed by methanol and NMP.

The incorporation of the radiometal into a conopeptide can beaccomplished at a Tyr residue for radio-iodine or will generally involvethe use of a chelate, specific to the particular metal, and a linkergroup to covalently attach the chelate to the conotoxin, i.e., a thebifunctional chelate approach. The design of useful chelates isdependent on the coordination requirements of the specific radiometal.DTPA, DOTA, P₂S₂—COOH BFCA requirement for kinetic TETA, NOTA are commonexamples. The requirement for kinetic stability of the metal complex isoften achieved through the use of multidentate chelate ligands with afunctionalized arm for covalent bonding to some part of the conantokinor γ-carboxyglutamate containing conopeptide, i.e., the lysine aminogroup. Techniques for chelating radioonuclides with proteins are wellknown in the art, such as demonstrated by interantional patentapplication publication No. WO 91/01144, incorporated herein byreference.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remingtoin's Pharmaceutical Scienices, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). Typically, an antagonistic amount of activeingredient will be admixed with a pharmaceutically acceptable carrier.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral,parenteral or intrathecally. For examples of delivery methods see U.S.Pat. No. 5,844,077, incorporated herein by reference.

“Pharmaceutical composition” means physically discrete coherent portionssuitable for medical administration. “Pharmaceutical composition indosage unit form” means physically discrete coherent units suitable formedical administration, each containing a daily dose or a multiple (upto four times) or a sub-multiple (down to a fortieth) of a daily dose ofthe active compound in association with a carrier and/or enclosed withinan envelope. Whether the composition contains a daily dose, or forexample, a half, a third or a quarter of a daily dose, will depend onwhether the phatiaceutical composition is to be administered once or,for example, twice, three times or four times a day, respectively.

The term “salt”, as used herein, denotes acidic and/or basic salts,formed with inorganic or organic acids and/or bases, preferably basicsalts. While pharmaceutically acceptable salts are preferred,particularly when employing the compounds of the invention asmedicaments, other salts find utility, for example, in processing thesecompounds, or where non-medicament-type uses are contemplated. Salts ofthese compounds may be prepared by art-recognized techniques.

Examples of such pharmaceutically acceptable salts include, but are notlimited to, inorganic and organic addition salts, such as hydrochloride,sulphates, nitrates or phosphates and acetates, trifluoroacetates,propionates, succinates, benzoates, citrates, tartrates, fumarates,maleates, methane-sulfonates, isothionates, theophylline acetates,salicylates, respectively, or the like. Lower alkyl quaternary ammoniumsalts and the like are suitable, as well.

As used herein, the tern “phannaceutically acceptable” carrier means anon-toxic, inert solid, semi-solid liquid filler, diluent, encapsulatingmaterial, formulation auxiliary of any type, or simply a sterile aqueousmedium, such as saline. Some examples of the materials that can serve aspharmaceutically acceptable carriers are sugars, such as lactose,glucose and sucrose, starches such as corn starch and potato starch,cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt,gelatin, talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol,polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate and ethyl laurate, agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcoholand phosphate buffer solutions, as well as other non-toxic compatiblesubstances used in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfateand magnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. Examples ofpharmaceutically acceptable antioxidants include, but are not limitedto, water soluble antioxidants such as ascorbic acid, cysteinehydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite,and the like; oil soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyaniisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, aloha-tocopherol and the like; and the metalchelating agents such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, ill which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques. The active agent can be encapsulated to make itstable to passage through the gastrointestinal tract while at the sametime allowing for passage across the blood brain barrier. See forexample, WO 96/11698.

For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

A variety of administration routes are available. The particular modeselected will depend of course, upon the particular drug selected, theseverity of the disease state being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, sublingual,topical, nasal, transdenial or parenteral routes. The term “parenteral”includes subcutaneous, intravenous, epidural, irrigation, intramuscular,release pumps, or infusion.

For example, administration of the active agent according to thisinvention may be achieved using any suitable delivery means, including:

(a) pump (see, e.g., Luer & Hatton (1993), Zimm et al. (1984) andEttinger et al. (1978));

(b), microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888;and 5,084,350);

(c) continuous release polymer implants (see, e.g., U.S. Pat. No.4,883,666);

(d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsW092/19195, WO 95/05452);

(e) naked or unencapsulated cell grafts to the CNS (see, e.g., U.S. Pat.Nos. 5,082,670 and 5,618,531);

(f) injection, either subcutaneously, intravenously, intra-arterially,intramuscularly, or to other suitable site; or

(g) oral administration, in capsule, liquid, tablet, pill, or prolongedrelease formulation.

In one embodiment of this invention, an active agent is delivereddirectly into the CNS, preferably to the brain ventricles, brainparenchyma, the intrathecal space or other suitable CNS location, mostpreferably intrathecally.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibodies or cell specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

The active agents, which are peptides, can also be administered in acell based delivery system in which a DNA sequence encoding an activeagent is introduced into cells designed for implantation in the body ofthe patient, especially in the spinal cord region. Suitable deliverysystems are described in U.S. Pat. No. 5,550,050 and published PCTApplication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.Suitable DNA sequences can be prepared synthetically for each activeagent on the basis of the known peptide sequences and disclosed DNAsequences.

The active agent is preferably administered in an therapeuticallyeffective amount. By a “therapeutically effective amount” or simply“effective amount” of an active compound is meant a sufficient amount ofthe compound to treat the desired condition at a reasonable benefit/riskratio applicable to any medical treatment. The actual amountadministered, and the rate and time-course of administration, willdepend on the nature and severity of the condition being treated.Prescription of treatment, e.g. decisions on dosage, timing, etc., iswithin the responsibility of general practitioners or spealists, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples oftechniques and protocols can be found in Remington's ParmaceuticalSciences.

Dosage may be adjusted appropriately to achieve desired levels, locallyor systemically, and depending on use as a diagnostic agent or atherapeutic agent. Typically the conopeptides of the present inventionexhibit their effect at a dosage range from about 0.001 mg/kg to about250 mg/kg, preferably from about 0.05 mg/kg to about 100 mg/kg of theactive ingredient, more preferably from a bout 0.1 mg/kg to about 75mg/kg, and most preferably from about 1.0 mg/kg to about 50 mg/kg. Asuitable dose can be administered in multiple sub-doses per day.Typically, a dose or sub-dose may contain from about 0.1 mg to about 500mg of the active ingredient per unit dosage form. A more preferreddosage will contain from about 0.5 mg to about 100 mg of activeingredient per unit dosage form. Dosages are generally initiated atlower levels and increased until desired effects are achieved. In theevent that the response in a subject is insufficient at such doses, evenhigher doses (or effective higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Continuous dosing over, for example 24 hours or multiple dosesper day are contemplated to achieve appropriate systemic levels ofcompounds.

Advantageously, the compositions are formulated as dosage units, eachunit being adapted to supply a fixed dose of active ingredients.Tablets, coated tablets, capsules, ampoules and suppositories areexamples of dosage forms according to the invention.

It is only necessary that the active ingredient constitute an effectiveamount, i.e., such that a suitable effective dosage will be consistentwith the dosage form employed in single or multiple unit doses. Theexact individual dosages, as well as daily dosages, are determinedaccording to standard medical principles under the direction of aphysician or veterinarian for use humans or animals.

The pharmaceutical compositions will generally contain from about 0.0001to 99 wt. %, preferably about 0.001 to 50 wt. %, more preferably about0.01 to 10 wt.% of the active ingredient by weight of the totalcomposition. In addition to the active agent, the pharmaceuticalcompositions and medicaments can also contain other pharmaceuticallyactive compounds. Examples of other pharmaceutically active compoundsinclude, but are not limited to, analgesic agents, cytokines andtherapeutic agents in all of the major areas of clinical medicine. Whenused with other pharmaceutically active compounds, the conopeptides ofthe present invention may be delivered in the form of drug cocktails. Acocktail is a mixture of any one of the compounds useful with thisinvention with another drug or agent. In this embodiment, a commonadministration vehicle (e.g., pill, tablet, implant, pump, injectablesolution, etc.) would contain both the instant composition incombination supplementary potentiating agent. The individual drugs ofthe cocktail are each administered in therapeutically effective amounts.A therapeutically effective amount will be determined by the parametersdescribed above; but, in any event, is that amount which establishes alevel of the drugs in the area of body where the drugs are required fora period of time which is effective in attaining the desired effects.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al.,1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988; Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Anilnal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzynies (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Metdods Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Imnmunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Riott, Essential Inmunology, 6th Edition,Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986).

EXAMPLES

The present invention is described by reference to the following, whichare offered by way of illustration and are not intended to limit theinvention in any manner. Standard techniques well known in the art orthe techniques specifically described below were utilized.

Example 1 Materials and Methods

Chelmical synthesis: Peptides were synthesized on a Rink amide resin,0.45 mmol/g [Fmoc-Cys(Trityl)-Wang; Novabiochem, San Diego, Calif.]usinig N-(9-fluorenyl)methoxycarboxyl chemistry and standard side chainprotection except on cysteine residues. Cysteine residues were protectedin pairs with either S-trityl on the first and third cysteines orS-acetamidomethyl on the second and fourth cysteines. Amino acidderivatives were from Advanced Chemtech (Louisville, Ky.). The peptideswere removed from the resin and precipitated, and a two-step oxidationprotocol was used to selectively fold the peptides as describedpreviously (Luo et al., 1999). Briefly, the first disulfide bridge wasclosed by dripping the peptide into an equal volume of 20 mM potassiumfeliicyanide and 0.1 M Tris, pH 7.5. The solution was allowed to reactfor 30 min, and the monocyclic peptide was purified by reverse-phaseHPLC. Simultaneous removal of the S-acetamidomethyl groups and closureof the second disulfide bridge was carried out by iodine oxidation. Themonocyclic peptide and HPLC eluent was dripped into an equal volume ofiodine (10 mM) in H₂ 0/trifluoroacetic acid/acetonitrile (78:2:20 byvolume) and allowed to react for 10 min. The reaction was terminated bythe addition of ascorbic acid diluted 20-fold with 0.1% trifluoroaceticacid and the bicyclic product purified by HPLC.

Mass Spectrometry: Measurements were performed at the Salk Institute forBiological Studies (San Diego, Calif.) under the direction of JeanRivier. Matrix-assisted laser desorption ionization time-of-flight massspectrometry and liquid secondary ionization mass spectrometry wereused.

Preparation of nACliR subunit cRNA: Attempts to express the rat nAChR α6subtype in Xenopus laevis oocytes consistently failed; that is, noACh-gated currents were detected. To improve functional expression, wecreated a chimeric receptor of the rat α6 and α3 subtypes. The chimericreceptor consists of amino acids 1 to 237 of the rat α6 subunit proteinlinked to amino acids 233 to 499 of the rat α3 subunit protein. Thechimeric junction is located at the paired-RR-residues immediatelypreceding the MI transmembrane segment of the α3 subunit. The resultingchimeric receptor represents the extracellular ligand-binding domain ofthe α6 subunit linked to membrane-spanning and intracellular segments ofthe α3 subunit. The α6/α3 cDNA was constructed by the introduction ofBspEI sites at the chimeric junction into the α6 and α3 cDNA sequencesusing mutagenic primers to introduce the restriction sites throughsilent codon changes. The α6 and α3 segments were generated bypolymerase chain reaction of rat brain cDNA using primers in the 5′ and3′ untranslated regions of the corresponding cDNAs along with theinternal muItagenic primers. The polymerase chain reaction products weredigested with BspEl and ligated to generate the chimeric construct. Thefinal chimeric construct was cloned and completely sequenced to confirmthe correct cDNA sequence. To further improve expression levels, all ofthe 5′ and 3′ untranslated regions of the nAChR cDNA were deleted, andthe chimeric construct was cloned into the X. laevis expression vectorpT7TS, placing X laevis globin 5′ and 3′ untranslated regions around thenAChR cDNA. The expression construct pT7TS/rα6α3 was transcribed with T7RNA polymerase to generate sense-strand RNA for oocyte expression.

Electrophysiology atid data analysis: Clones of rat nAChR subunits wereused to produce cRNA for injection into X laevis oocytes as describedpreviously (Cartier et al., 1996). The rat α6 and β3 subunits were agenerous gift from S. Heinemann (Salk Institute, San Diego, Calif.)(Deneris et al., 1989). To express nAChRs in oocytes, 5 ng of each nAClRsubunit was injected. In the case of α6β4, 50 ng of each subunit wasinjected because of absent expression when using 5 ng of cRNA. Likewise,20 ng was used for the α6/α3β2 combination that expresses poorly withoutthe β3 subunit. A 30-μl cylindrical oocyte recording chamber fabricatedfrom Sylgard was gravity-perfused with ND96A (96.0 mM NaCl, 2.0 mM KCl,1.8 mM CaCl₂, 1.0 mM MgCl₂, 1 μM atropine, and 5 mM HEPES, pH 7.1-7.5)at a rate of ˜2 ml/min (Luo et al., 1998). All toxin solutions alsocontained 0.1 mg/ml bovine serum albumin to reduce nonspecificadsorption of peptide. Toxin was preapplied for 5 min. ACh-gatedcurrents were obtained with a 2-electrode voltage-clamp amplifier (modelOC-725B; Warner Instrument, Hamden, CT), and data were captured asdescribed previously (Luo et al., 1998). The membrane potential of theoocytes was clamped at −70 mV. To apply a pulse of ACh to the oocytes,the perfusion fluid was switched to one containing ACh for 1 s. This wasdone automatically at intervals of 1 to 5 min. The shortest timeinterval was chosen such that reproducible control responses wereobtained with no observable desensitization. The concentration of AChwas 10 μM for trials with α1β1δεand 100 μM for all other nAChRs. Toxinwas bath-applied for 5 min, followed by a pulse of ACh. Thereafter,toxin was washed away, and subsequent ACh pulses were given every 1 min,unless otherwise indicated. All ACh pulses contain no toxin, for it wasassumed that little if any bound toxin washed away in the brief time(less than the 2 s it takes for the responses to peak). In our recordingchamber, the bolus of ACh does not project directly at the oocyte butrather enters tangentially, swirls, and mixes with the bath solution.The volume of entering ACh is such that the toxin concentration remainsat a level >⁵⁰% of that originally in the bath until the ACh responsehas peaked (<2 s). When longer than 5 min of toxin application wasneeded to reach maximum block, toxin was applied by continuous perfusionto the oocytes as described previously (Luo et al., 1994), except thatACh was applied once every 2 min.

The average peak amplitude of three control responses just precedingexposure to toxin was used to normalize the amplitude of each testresponse to obtain a “% response” or “% block” . Each data point of adose-response curve represents the average value± S.E. of measurementsfrom at least three oocytes. Dose-response curves were fit to theequation %response=100/{1+([toxin]/IC₅₀)^(nH) }, where n_(H) is the Hillslope detenrined with Prism software (Graph-Pad Software, San Diego,Calif.) on an Macintosh (Apple Computers, Cupertino, Calif.). For threeor fewer data points, nH was set to 1.0.

Membrane preparation: Mice were killed by cervical dislocation. Brainswere removed from the skulls and dissected on an ice-cold platform.Membranes containing [¹²⁵I]α-conotoxin MII binding sites were preparedfrom pooled olfactory tubercles, striatum, and superior colliculus.Samples were homogenized in 2× physiological buffer (288 mM NaCl; 3 mMKCl; 4 mM CaCl₂; 2 mM MgSO₄; and 40 mM HEPES, pH 7.5; 22° C.) using aglass-polytetrafluoroethyle tissue grinder. Homogenates were thentreated with phenylmethylsulfoniyl fluoride (final concentration, 1 mM;15 min at 22° C.) to inactivate endogenous serine proteases beforecentrifugation (20,000g for 20 min at 4° C.). Pellets were washed twiceby homogenization in distilled deionized waterglass-polytetrafluoroethylene tissue grinder, 4° C.) and centrifugation(20,000g for 20 min at 4° C.). Pooled tissue from a single mouseprovided sufficient material for a single 96-well fonrat assay.

Inhibition of [¹²⁵I] α-conotoxin MII binding: Inhibition of [¹²⁵I]α-conotoxin MII binding to mouse brain membranes was performed using amodified version of the 96-well plate procedure described previously(Whiteaker et al., 2000a). Assays were performed in triplicate using1.2-ml siliconized polypropylene tubes arranged in a 96-well formiat.Membrane pellets were resuspended into distilled deionized water. Total(no drug) and nonspecific (with 1 μM epibatidine) binding determinationswere included in each experiment for each drug dilution series. Initialincubations proceeded for 3 h at 22° C. in 1× protease inhibitor buffer[1× physiological buffer supplemented with bovine serum albumin (0.1%w/v), 5 mM EDTA, 5 mM EGTA, and 10 μg/ml each of aprotinin, leupeptintrifluoroacetate, and pepstatin A]. Each tube contained 10 μl ofmembrane preparation, 10 μl of competing ligand (or nonspecific or totaldeterminations) in 1× protease inhibitor buffer, and 10 μl of[¹²⁵I]α-conotoxill MII (1.5 nM in 2× protease inhibitor buffer, giving afinal assay radioligand concentration of 0.5 nM). After incubation, eachtube was diluted with 1 ml of physiological buffer plus 0.1% (w/v)bovine serum albumin. Tubes were then incubated for a further 4 mil at22° C. to reduce nonspecific binding to the membrane preparation. Thebinding reactions were then terminated by filtration onto a singlethickness of GF/F filter paper (Whatman, Clifton, N.J.) using a cellharvester (Inotech Biosystems, Rockville, Md.). The filters wereincubated previously for 15 min with 5% dried skim milk to reducenonspecific binding. Assays were washed with four changes ofphysiological buffer supplemented with bovine serum albumin (0.1% w/v).Washes were performed at 30-s intervals, with each lasting approximately5 s. All filtration and collection steps were perfonred at 4° C. Boundligand was quantified for each filter disc by gamma counting using aCobra II counter (≈85% efficiency) (PerkinElmer Life and AnalyticalSciences, Boston, Mass.).

Calculations: Data from individual [¹²⁵I]α-conotoxin MII inhibitionbinding experiments were processed using a single-site fit using thenonlinear least-squares fitting algorithm of GraphPad Prism. Values ofK_(i) were derived for each experiment by the method described by Chengand Prusoff (1973), K_(i)=IC₅₀/l+(L/K_(D)), where K_(i) for [¹²⁵I]α-conotoxin is 0.32 nM.

Example 2 Results

Pepticle Syiithesis: The sequence of native α-conotoxin MII isGCCSNPVCHLEHSNLC. Peptide analogs were synthesized by substituting oneor more residues with alanine. These peptides are named according to theresidue(s) substituted; for example, MII[E11A] has the glutamic acid inposition 11 substituted with alanine. Cysteine residues wereorthogonally protected to direct the formation of disulfide bonds in theconfiguration found in α-conotoxin MII, that is cysteine 1 to cysteine 3and cysteile 2 to cysteine 4. The first and third cysteine residues wereprotected with acid-labile groups that were removed first after acleavage from the resin; ferricyanide was used to close the firstdisulfide bridge. The monocyclic peptides were purified by reverse-phaseHPLC. Then the acid-stable acetamidomethyl groups were removed from thesecond and fourth cysteines by iodine oxidation that also closed thesecond disulfide bridge. The fully folded peptides were again purifiedby HPLC. Mass spectrometry was used to confirm synthesis. The observedmolecular mass for each peptide was within 0.1 Da of the expected mass.

Peptide Effects on α6 and α3 nAChRs: Injection of rat α6 subunits intooocytes either alone or in combination with β2 and/or β3 subunits yieldsfew or no functional nAChRs. Using a previously reported strategy forhuman α6 (Kuryatov et al., 2000), we joined the extracellular domain ofthe rat α6 subunit to the transmembranie and intracellular portion ofthe closely related rat α3 subunit. Alanine analogs were then testedagainst α3β2 or α6/α3β2β3 subunit combinations heterologously expressedin oocytes. The β3 subunit was used with α6/α3β2, for without it therewas generally little or no functional expression. In addition, β3 isassociated with native α6β2*-containing nAChRs (Zoli et al., 2002; Cuiet al., 2003). Results are shown in FIG. 1 and Table 1. TABLE 1 Activityof alanine-substituted MII analogs IC₅₀ Rat α3β2 Rat α6/α3β2β3 Toxin nMRatio^(a) MII 2.18 (1.24-3.81) 0.39 (0.281-0.548) 5.59 MII[S4A] 15.8(7.03-35.3) 0.733 (0.513-1.05) 21.56 MII[N5A] >10,000 793(566-1110) >12.6 MII[P6A]   4,420 (1880-10,400) 253 (172-372) 17.5MII[V7A] 4.46 (3.28-6.05) 10.6 (8.01-14.0) 0.421 MII[H9A] 59.0(44.1-78.9) 0.790 (0.558-1.12) 74.7 MII[L10A]  1.47 (0.642-3.38) 0.482(0.232-1.00) 3.05 MII[E11A] 8.72 (6.84-11.1) 0.160 (0.135-0.189) 54.5MII[H12A] 4,660 (2420-9000) 604 (256-1420) 7.72 MII[S13A] 2.54(1.92-3.35) 0.659 (0.450-0.966) 3.85 MII[N14A] 25.7 (17.0-38.9) 1.06(0.742-1.52) 24.2 MII[L15A] 34.1 (19.4-59.9) 0.917 (0.657-1.28) 37.2^(a)IC₅₀ α3β2/IC₅₀ α6/α3/β2β3.Numbers in parentheses are 95% confidence intervals.

Substitution of alanine for Asn5, Pro6, or His 12 resulted insubstantially decreased activity compared with native MII at both a 6*and a 3* NAChRs, whereas substitution for Val7 had the most pronouncedeffect on the α6/α3,β2β3 nAChR. Substitution for Ser4, His9, Leu10,Glu11, Ser13, Asn14, and Leu15 had only modest effects on α6/α3β2, β3;however, mutations a Ser4, His9, Glu11, Asn14, and Leu15 resulted insubstantially lower activity on α3, β2 nAChRs. Thus, these mutations areanalogs that preferentially block α6/α3β2β3 versus α3β2 nAChRs. We notethat for certain analogs, including S4A, L10A, E11A, S13A, and N14A, thet_(1/2) for recovery from toxin block of α6/α3β2β3 nAChLRs was long (>25min). For these analogs, 10- to 15-min toxin incubations were used toachieve maximum block at 10 nM concentration, and 20- to 35-minincubations were used to achieve maximum block at I nM concentration. Aslow off-rate but similar affinity to other analogs implies that theanalogs with a slow off-rate also have slower on-rates and thus the needfor longer application times. MII[H9A] and MII[L15A] failed to blockα4β2 nAChRs; at 10 μM peptide concentration, the ACh-evoked current was105.8±2.4 and 102.3±5.3% of control, respectively (data from sixoocytes).

Selectivity; of MII[E11A]: The single alanine substitution MIl[E11A] has˜50-fold preference for α6/α3β2β3 versus α3β2 nAChRs and seems to be themost potent analog on α6/α3β2β3 nAChRs. We therefore tested its effectson additional nAChR subtypes. The apparent on-rate for α6/α3β4 nAChRs isslow; at concentrations of toxin ≦10 nM, 60 to 70 min of toxinapplication was required to reach a steady-state level of nAChR block.Concentration-response curves are shown in FIG. 2 and IC₅₀ values areshown in Table 2. TABLE 2 Activity of MII[E11A] IC₅₀ 95% Confidence nMInterval Ratio^(a) α2β2 >10,000 >62,500 α2β4 >10,000 >62,500 α3β2 8.726.84-11.1 54.5 α3β4 2100 1330-3310 13,100 α4β4 >10,000 >62,500 α6/α3β20.154 0.134-0.178 0.962 α6/α3β2β3 0.160 0.135-0.189 1.00 α6/α3β4 6.444.33-9.57 40.3 α7 1051  (731-1510) 6,570^(a)nAChR subtype IC₅₀/α6/α3β2β3 IC₅₀

Double Mutants: A series of double alanine-substituted mutations wasalso constructed. These mutations were tested with respect to theiractivity at α6/α3β2β3 and α3β2 nAChRs. As seen in Table 3, each of thesedouble mutants preferentially blocks the α6/α3β2β3 receptor versus theα3β2 receptor. The IC₅₀ of the MII[H9A;L15A] analog was approximately2000-fold lower for α6/α3β2β3 versus α3β2, and this analog was selectedfor further characterization (FIG. 3). TABLE 3 Activity of doublysubstituted MII analogs IC₅₀ Rat α3β2 Rat α6/α3β2β3 Toxin nM Ratio^(a)MII[S4A; H9A] 207 (156-274)  1.97 (1.31-2.97) 105 MII[H9A; L15A]  4850(3540-6630) 2.40 (1.68-3.43) 2020 MII[L10A; 17.2 (8.11-36.6) 1.80(1.26-2.56) 9.56 L15A] MII[E11A; 50.1 (41.4-60.6)  0.415 (0.223-0.772)121 L15A]^(a)IC₅₀ α3β2/IC₅₀ α6/α3/β2β3.Numbers in parentheses are 95% confidence intervals.

Kinetics of Block by MII[H9A;L 15A]: α-Conotoxin MII is slowlyreversible α3β2 nAChRs and very slowly reversible on α6/α3β2β3 nAChRs(FIG. 4). Substitution of Ala for His9 or Leu15 leads to more rapidrecovery from block for both receptor subtypes. In the case of thedouble mutant MII[H9A;L15A] recovery from toxin block is rapid. Themagnitude of the change of recovery rate is greater than the magnitudeof change in IC₅₀ at the α6/α3β2β3 receptor. This implies that changesin the peptide that lead to a rapid off-rate also lead to a fasteron-rate of binding. We note that α-conotoxini GIC, a more rapidlyreversible homolog of α-conotoxin MII, also has an alaninie rather thanthe histidinie found in position 9 of α-MII (McIntosh et al., 2002).Activity of MII[H9A;L15A] on Other nAChR Subtypes: MII[H9A;L15A] hashighest affinity for the α6/α3β2β3 subunit combination and ˜100-foldless activity on the α6/α3β4 combination (FIG. 3). MII[H9A;L15A] has lowor no activity on the remaining neuronal subunit combinations tested,including α2β2, α2β4, α3β4, α4β2, α4β4, and α7 (FIG. 5 and Table 4).Thus MII[H9A;L15A] selectively blocks α6* nAChRs, with preference forthe α6/α3β2β3versus α6/α3β4 subunit combination. TABLE 4 Effects ofMII[H9A; L15A] Concentration of MII[H9A; L15A] Receptor 10 μM 1 μM Rα2β2 102 ± 1.4 103 ± 1.9 Rα2β4 96.7 ± 3.2 95.9 ± 2.3  Rα4β2 98.5 ± 4.4 101 ±2.5 Rα4β4 99.0 ± 4.0 100 ± 5.6 Rα7 61.4 ± 2.3 100 ± 1.5Values are the percentage of control ± S.E.M.

Effect of β3 Subunit: Occasional expression of α6/α3β2 was seen withoutcoinjection of the β3 subunit. MII[H9A; L15A] blocked α6/α3β2 nAChRswith an IC₅₀ of 8.21 (6.36-10.6) nM compared with 2.4 nM (1.68-3.43) forα6/α3β2β3. As indicated above (FIG. 2 and Table MII[E11A] blockedα6/α3β2 nAChRs with an IC₅₀ of 0.154 nM (0.134-0.178) compared with 0.16nM (0.135-0.189) on α6/α3β2β3 NAChRs. Numbers in parentheses are 95%confidence intervals.

Activity of Analogs at Native Moutse Brain nAChRs: Aconcentration-response analysis was performed on four of the analogswith α6/α3* versus α3* selectivity—MII[H9A], MII[E11A], MII[L15A], andMII[H9A;L15A]—using inhibition of [¹²⁵I]α-conotoxin MII binding(Whiteaker et al., 2000b) to mouse brain homogenates. Results are shownin FIG. 6. The values obtained for these analogs correlate well withvalues obtained on α6/α3β2β3 rather than α3β2 nAChRs as expressed in Xlaevis oocytes (Table 5). TABLE 5 α-Conotoxin MII and analogs IC₅₀ K_(i)Mouse IC₅₀ Rat α3β2 Rat α6/α3β2β3 CNS α-MII Site Peptide nM α-MII  2.2(1.2-3.8)  0.39 (0.28-0.55) 0.22 (0.20-0.25) MII[H9A] 59 (44-79) 0.79(0.56-1.1) 1.1 (0.84-1.6) MII   8.7 (6.8-11.1)  0.16 (0.13-0.19) 0.27(0.19-0.37) [E11A] MII 34 (19-60) 0.92 (0.65-1.3) 0.30 (0.21-0.45)[L15A] MII[H9A;  4800 (3500-6600) 2.4 (1.7-3.4) 3.3 (2.5-4.3)  L15A]Rat nAChRs were heterologously expressed in oocytes, and functionalblock of ACh-induced current was measured. Radioiodinated α-conotoxinMII was used with mouse brain homogenates to examine the competitionbinding of the indicated peptides. See FIGS. 1, 2, 3, and 6.Numbers in parentheses are 95% confidence intervals.

Example 3 Discussion

Although the sequence of the coding region for the α6 gene has beenknown for many years (Lamar et al., 1990), its functional significancehas been challenging to elucidate because of difficulties inheterologously expressing α6 and because of a lack of subtype-specificligands. Indeed, originally it was not entirely certain that the α6 geneencoded a nicotinic receptor subunit. The α6 subunit has relativelydiscrete localization, with expression in cataclholaminergic nucleiincluding the locus coeruleus, the ventral tegmental area, and thesubstantia nigra (Le Novèere et al., 1996; Goldner et al., 1997; Han etal., 2000; Quik et al., 2000; Azam et al., 2002). It is also found intrigeminal ganglion and olfactory bulb (Keiger and Walker, 2000). Inaddition, α6 complexes have been reported in chick retina (Vailati etal., 1999). The α6 mRNA expression pattern overlaps extensively withthat of the α3 subunit, leading to initial confusioni over thecomposition of [¹²⁵ I ]α-conotoxin MII-binding nAChRs (Whiteaker et al.,2000b).

Subunit-specific antibodies have been used to immunoprecipitate α6*receptors from chick retina. When reconstituted in lipid bilayers, thesereceptors formed cationic channels characteristic of nAChRs, thusestablishing a functional role for native α6* nAChRs (Vailati et al.,1999). Antibodies have also been used recently to demonstrate thepresence of α6β2* nAChRs in striatal dopaminergic terminals in rat. β03and/or α4 subunits are also present in a proportion of these iiAChRs(Zoli et al., 2002). Subunit knockout mice suggest that thehigh-affinity binding site of [¹²⁵I]α-conotoxin MII is predominatelycomposed of α6* rather than α3* nAChRs (Champtiatix et al., 2002;Whiteaker et al., 2002). It has been hypothesized recently that putativeα6* nAChRs in the striatum may participate in the pathophysiology ofParkinson's disease, a neurodegenerative disorder characterized byprogressive loss of dopamine neurons. Treatment of primates with1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (a dopaminergic neurotoxin)leads to selective decline of putative α6β2* nAChRs (Quik et al., 2001;Kulak et al., 2002). Thus, there is a significant need for ligands thatselectively act at α6* nAChRs.

We demonstrate in this report that certain analogs of α-conotoxin MIIexhibit preferential loss of activity at α3β2 versus α6/α3β2β3 nAClRs.Additionally, at concentrations tested, the MII[H9A;L15A] analog haslittle or no activity at α2*, α4*, or 7* nAChRs. Indeed, MII[H9A;L15A]is the most selective α6 ligand thus far reported.

A number of native α-conotoxins have been characterized that targetvarious subtypes of nAChRs. Despite their differences in primarysequence, NMR and X-ray crystallography studies show a high conservationof the structural peptide backbone (Hu et al., 1996, 1997, 1998; Shon etal., 1997; Hill et al., 1998; Cho et al., 2000; Park et al., 2001; Nickeet al., 2003). It seems that this backbone serves as a scaffold thatpresents a variety of amino acid side chains leading to differences inspecificity. In the present study, we have systematically replaced thenoncysteine residues of α-conotoxin MII with alaninie. The predominanteffect is to preferentially decrease activity at the α3β2 receptorrelative to the α6/α3Γ2(±β3) subunit combination.

In an attempt to further increase selectivity, double mutations wereconstructed from the more selective single mutation analogs. Each ofthese double mutants retalns a low nanomolar IC₅₀ for the α6/α3β2β3nAChR (Table 3). It is particularly noteworthy that the native MIIpeptide potently blocks both α6/α3β2β3 and α3β2 NAChRs, whereas theMII[H9A;L15A] analog discriminates between these nAChRs by three ordersof magnitude. This discrimination is caused by differences in theextracellular region of the αsubunit because the transmembrane andintracellular portions of the chimeric α6/α3 and α3 subunits areidentical. Also, the addition of the β3 subunit to α6/α3β2 nAChRs hasonly a 3.4-fold effect on MII[H9A,L15A] block. MII[E11A] alsopreferentially blocks α6/α3β2 versus α3β2 nAChRs, again implicating theextracellular portion of the α6 subunit. Furthermore, coexpression ofthe β3 subunit with the α6/α3 and 62 12 subunits had no effect on theIC₅₀ of MII[E11A]. However, the presence of a β2 versus β4 subunit doesseem to influence peptide affinity. MII[E11lA] preferentially blocksα6/α3β2 versus α6/α3β4 nAChRs and preferentially blocks α3β2 versus α3β4nAChRs.

We used cloned rat receptor subunits heterologously expressed in Xlaevis oocytes to examine the differences between α3* and x6* nAChRs.Although difficult, occasional expression of α6 with either β2 or β4subunits has been described. This expression is enhanced with theaddition of the β3 subunit (Kuryatov et al., 2000+). Improved efficiencyof expression has been achieved by combining the extracellular (putativeligand binding) domain of α6 with the remaining portion of either the α3or α4 subunit (Kuryatov et al., 2000). We have exploited this techniqueto screen analogs of α6-conotoxin MII. It is possible that there areimportant differences between this chimeric receptor expressed inoocytes and native nACliRs. To assess this, the α-conotoxin MII analogswere also tested in a radioligand binding assay using native nAChRpopulations. As can be seen in Table 5, the analogs that potently blockthe rat α6/α3β2β3 nAChR heterologously expressed in oocytes alsopotently block the native mouse striatal nAChR bound by radiolabeledMII. This native receptor has been shown in previous studies to containα6 (rather than α3) and β2 subunits (Champtiaux et al., 2002; Whiteakeret al., 2002; Zoli et al., 2002). Thus, the analogs have high affinityfor both native and heterologously expressed α6β2* nAChRs. The H9A;L15Aanalog of MII also has a relatively high IC₅₀ for other NAChRs,including α2β2, α2β4, α3β2, α3β2, α4β2, α4β2, α4β4, and α7. Thus, thispeptide represents a novel selective probe for discriminating amongnumerous nAChR subunit combinations.

The precise mechanism by which the [H9A] and [L15A] mutations cause aselective loss of affinity at α3β2 relative to α6/α3β2* nAChRs is notaddressed by these studies. It has been determined that Lys185 andIle188 of the α3 subunit are critically important for α-conotoxin MIIbinding to α3β2 nAChRs (Harvey et al., 1997), and these residues areconserved between the α3 and α6 subunits. The most facile explanation ofthe results presented here is that the crucial interactions betweenα-conotoxin MII and the α6 subunit may occur at other subunit sidechains. Interestingly, both α-conotoxin PnIA and α-conotoxin MIIinteract with Ile188 but differ in other important interactions with theα3 subunit. α3 subunit Lys 185 is not essential to α-conotoxin PnIAbinding, whereas Pro182 and Gln198 are (Everhart et al., 2003). Perhapssignificantly, the latter two {grave over (r)}esidues are not conservedbetween the α3 and α6 subunit. Because all of the above residues arefound in the putative “C” loop of the α-subunit, it seems possible thatinteraction in this region may be of particular importance. However,several examples indicate that a more complex explanation may be needed.For instance, α-conotoxin PnIA and its derivative α-conotoxin PnIA[A10L]stabilize different states of the same nAChR (.Hogg et al., 2003),presumably by interacting with different sets of subunit residues,whereas α-conotoxin MI has been shown to interact in a differentorientation with the same α subunit residues, depending on whether it isbinding at an α/γ or α/δinterface (Sugiyama et al., 1998). These and aseries of mutant-cycle analysis studies (Quiram et al., 1999, 2000; Brenand Sine, 2000) have indicated that toxin/channel interactions may beanchored by a small number of relatively strong interactions andsupported by a large number of weaker interactions that stronglydetermine subtype selectivity (Rogers et al., 2000). If this moremultifaceted model is correct, the maintenance of affinity betweenα-conotoxin MII[H9A;L15A] and the α6/α3β2* nAChR may reflect either amore prominent role of the “supporting” interactions with the nativetoxin than is seen for α3β2, which is retained after alteration of theHis9 and Leu15 side chains.

Alternatively, the orientation of the toxin within the binding pocketmay shift after substitution at the His9 and Leu15 positions, but thestructure of the α6/α3 binding pocket may be better able to accommodatethe new positioning than its α3 counterpart. The fact that several ofthe alanine mutants exhibit affinities similar to each other and nativeα-conotoxin MII but have radically different binding kinetics reinforcesthe idea that different interactions may stabilize the nAChR/toxincomplex in each case. It seems likely that an accurate understanding ofhow the [H9A] and [L15A] mutations produce selectivity between α3β2 andα6/α3β2* nAChRs will require the performance of a comprehensive set ofdouble mutant-cycle analyses.

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, the described embodiments are illustrative andshould not be construed as restrictive.

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1. An isolated conopeptide selected from the group consisting of: (a) aconopeptide described herein, with the proviso that the conopeptide isnot α-contoxin MII (SEQ ID NO:1); (b) a permutant of (a); (b) aderivative of (a) or (b); (c) a physiologially acceptable salt of (a),(b) or (c).
 2. A pharmaceutical composition comprising the conopeptideof claim 1 and a pharmaceutically acceptable carrier.
 3. A method fortreating a disorder associated with α6-containing nAChRs comprisingadministering a therapeutically effective amount of the conopeptide ofclaim 1 to an individual exhibiting said disorder or in risk ofexhibiting said disorder.
 4. A method for treating a disorder associatedwith α6-containing nAChRs comprising administering a therapeuticallyeffective amount of the phanraceutical composition of claim 2 to anindividual exhibiting said disorder or in risk of exhibiting saiddisorder.
 5. A method of identifying compounds that mimic thetherapeutic activity of the conopeptide of claim 1, comprising the stepsof: (a) conducting a biological assay on a test compound to determinethe therapeutic activity; and (b) comparing the results obtained fromthe biological assay of the test compound to the results obtained fromthe biological assay of the conopeptide of claim
 1. 6. A method fordistinguishing α3-containing nAChRs and α6-containing nAChRs whichcomprises contacting a conopeptide of claim 1 with a nAChR, determiningthe response of the nAChR to the conopeptide and correlating theresponse with the known response of the conopeptide with α3-containingnAChRs and α6-containing NAChRs.